Báo cáo khoa học: cAMP response element-binding protein (CREB) is imported into mitochondria and promotes protein synthesis docx

cAMP response element-binding protein (CREB) is imported
into mitochondria and promotes protein synthesis
Domenico De Rasmo
1
, Anna Signorile
1
, Emilio Roca
1
and Sergio Papa
1,2
1 Department of Medical Biochemistry, Biology and Physics (DIBIFIM), University of Bari, Italy
2 Institute of Biomembranes and Bioenergetics (IBBE), Consiglio Nazionale delle Ricerche, Bari, Italy
Introduction
The cAMP response element-binding protein (CREB)
is a ubiquitous transcription factor in the higher
eukaryotes that recognizes the DNA consensus
sequence TGACGTCA, the cAMP response element
(CRE) in gene promoters [1,2].
Phosphorylation of CREB by cAMP-dependent pro-
tein kinase (protein kinase A; PKA), as well as by
Ca
2+
-dependent and other protein kinases, in
response to different cellular signals, promotes tran-
scription of CRE-regulated genes [1–4]. Activation of
the expression of nuclear CRE-regulated genes has
been shown to be involved in a variety of cellular pro-
cesses, including apoptosis [5,6], oxidative stress [7],
neuronal growth, and plasticity [5,8]. In yeast, cAMP
was found to reverse the glucose repression of mito-
chondriogenesis [9] and to activate the expression of

mitochondrial genes [10,11] and nuclear genes [12,13]
of respiratory chain proteins. In Saccharomyces cerere-
visiae, where the RAS ⁄ cAMP ⁄ PKA system appears to
be involved in regulation of the biogenesis of the
oxidative phosphorylation system [13], a probable cis-
regulatory element on mtDNA, responsible for cAMP-
mediated transcription, was identified [11]. In yeast
and mammalian cells, the cAMP cascade is involved in
the regulation of mitochondrial dynamics [14] and
bioenergetics [15–17].
In 1999, findings were presented [18] indicating that
CREB is localized in the inner mitochondrial compart-
ment as well as in the nucleus. These observations,
based on the use of CREB and phospho-CREB anti-
Keywords
cAMP cascade; complex I; CREB;
mitochondrial protein synthesis; PKA
Correspondence
S. Papa, Department of Medical
Biochemistry, Biology and Physics,
University of Bari, Policlinico,
P.zza G. Cesare, 70124 Bari, Italy
Fax: +39 080 5448538
Tel: +39 080 5448540
E-mail:
(Received 9 March 2009, revised 26 May
2009, accepted 4 June 2009)
doi:10.1111/j.1742-4658.2009.07133.x
The cAMP response element-binding protein (CREB) is a ubiquitous
transcription factor in the higher eukaryotes that, once phosphorylated,

promotes transcription of cAMP response element-regulated genes. We
have studied the mitochondrial import of CREB and its effect on the
expression of mtDNA-encoded proteins. [
35
S]Methionine-labelled CREB,
synthesized in vitro in the Rabbit Reticulocyte Lysate system using a con-
struct of the human cDNA, was imported into the matrix of isolated rat
liver mitochondria by a membrane potential and TOM complex-dependent
process. The imported CREB caused cAMP-dependent promotion of the
synthesis of mitochondrially encoded subunits of oxidative phosphorylation
enzyme complexes. Thus, CREB moves from the cytosol to mitochondria,
in addition to the nucleus, and, when phosphorylated by cAMP-dependent
protein kinase, promotes the expression of mitochondrial genes.
Abbreviations
ADU, arbitrary densitometric units; AKAP, A kinase anchoring protein; CAP, chloramphenicol; cPKA, catalytic subunit of cAMP dependent
protein kinase; CRE, cAMP response element; CREB, cAMP response element-binding protein; db-cAMP, dibutyryl cAMP; IBMX,
isobutylmethylxanthine; PKA, cAMP-dependent protein kinase (protein kinase A); RRL, Rabbit Reticulocyte Lysate.
FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4325
bodies, as well as on mtDNA mobility shift assays,
were confirmed by Lee et al. [19] and Ryu et al. [20].
Lee et al. also showed that binding of CREB to CRE
sequences in the D-loop of mtDNA increased the tran-
script levels of the ND2, ND5 and ND6 mitochondrial
genes of complex I of the respiratory chain. Ryu et al.
[20] found that activation by the antioxidant iron che-
lator deferoxamine of PKA, localized in the mitochon-
drial matrix [21], promoted CREB binding to the
mtDNA D-loop. The presence in mitochondria of the
CREB factor and, in particular, the use of phospho-
CREB antibody to detect this transcription factor in

mitochondria were, however, questioned by Platenik
et al. [22]. These authors showed that commercially
available antibodies for phospho-CREB crossreact
with the E1 a-subunit of mitochondrial pyruvate dehy-
drogenase. Because of the relatively high abundance in
mitochondria of pyruvate dehydrogenase, this crossre-
activity might overwhelm the reaction of the antibody
with phospho-CREB and produce false-positive
results. The general physiological relevance of a possi-
ble role of CREB and PKA in providing a regulatory
mechanism in the expression of mitochondrially
encoded proteins of the respiratory chain prompted us
to investigate the mitochondrial import of the CREB
protein, synthesized in vitro, and its effect on the
expression of mitochondrial genes. The results
unequivocally show that exogenous CREB is imported
into isolated rat liver mitochondria and causes a
marked, cAMP-dependent, stimulation of the expres-
sion of the mitochondrially encoded subunits of oxida-
tive phosphorylation complexes.
Results
[
35
S]Methionine-labelled CREB is imported into
isolated mitochondria
Mitochondrial import of CREB was investigated by
incubating freshly isolated, intact rat liver mitochon-
dria with the protein synthesized in the Rabbit Reti-
culocyte Lysate (RRL) system with [
35

S]methionine.
Figure 1A,B shows time-dependent mitochondrial
uptake of the radioactive CREB, which was largely
resistant to trypsin digestion unless mitochondria were
dissolved by Triton X-100 (Fig. 1C, lane 3). The
amount of mitochondrial proteins and mitochondrial
integrity were checked by immunodetection of the
39 kDa subunit of the inner membrane complex I, in
the absence and in the presence of trypsin. Mitochon-
drial uptake of CREB was promoted by the mitochon-
drial membrane potential, as shown by its inhibition
by valinomycin (Fig. 1C, lane 4). The residual radio-
active CREB detected in the mitochondrial pellet in
the presence of valinomycin represents the amount of
protein bound at the mitochondrial outer surface, as
shown by its complete digestion by trypsin (Fig. 1C,
lane 5). When mitochondria were pretreated with
A
B
C
400
300
200
100
0
0 10203040506070
Fig. 1. Import into isolated mitochondria of [
35
S]methionine-labelled
CREB. [

35
S]Methionine-labelled CREB, synthesized in the RRL
translation system, was added to isolated rat liver mitochondria.
(A, C) Autoradiograms of SDS ⁄ PAGE slabs of the mitochondrial
pellet. The RRL gel slab on the left of (A) is an autoradiogram of an
amount of the radioactive CREB synthesis mixture corresponding
to half of the amount added to mitochondria for the import assay.
No precipitable aggregate of radioactive CREB was detectable after
centrifugation of the RRL CREB synthesis translation mixture in the
absence of added mitochondria. Where indicated, mitochondria,
after completion of the import incubation, were treated, before
pelletting, with trypsin (1 lg per 50 lg of mitochondrial protein) for
35 min at 0 °C. (C) Import incubation for 60 min: in lane 3, mito-
chondria were treated with trypsin in the presence of 0.2% Triton
X-100; in lanes 4 and 5, valinomycin (Val) (0.1 lg per mg of
mitochondrial protein) was present during the import incubation.
(B) Mean values in arbitrary densitometric units (ADU) (three sepa-
rate experiments) of the trypsin-resistant [
35
S]methionine-labelled
CREB radioactivity, detected in the mitochondrial pellet, plotted as
a function of the import incubation time. The SDS ⁄ PAGE slabs
were also blotted with an antibody against the 39 kDa subunit of
complex I. See Experimental procedures and [24] for further
details.
CREB and mitochondrial protein synthesis D. De Rasmo et al.
4326 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS
trypsin, before the import assay of radioactive CREB
(Fig. 2A, lanes 3 and 4), the amount of imported
CREB, resistant to trypsin treatment after completion

of the uptake, and that of externally bound CREB,
digested by trypsin, were both reduced. This indicates
that mitochondrial binding and import of CREB
involve surface components of the outer membrane
import complex. Addition to the mitochondrial import
mixture of an antibody against Tom20, an outer
membrane receptor of the mitochondrial import sys-
tem [23], reduced the binding of [
35
S]methionine-
labelled CREB to the mitochondrial surface (amount
digested by trypsin) and the accumulation in mito-
chondria (trypsin-resistant amount) (Fig. 2B, lanes 5
and 6). No significant effect was exerted on CREB
binding and import by an antibody against Tom70
(Fig. 2B, lanes 3 and 4).
The dependence of CREB uptake on Tom20 and
membrane potential indicates that the protein reaches
the inner mitochondrial compartment. Submitochon-
drial localization of the imported CREB was directly
verified by separation of mitochondrial subfractions
(Fig. 3). After the import incubation and trypsin treat-
ment of mitochondria, organelles were swollen in a
hypo-osmotic medium to eliminate CREB bound at
the surface. Residual mitochondria and mitoplasts,
deprived of the outer membrane and of residual super-
ficially bound radioactive CREB, were disrupted by
sonication, and the inner membrane fraction was sepa-
rated from the matrix content. The radioactive CREB
of the mitoplast fraction, still resistant to trypsin in

this fraction, was recovered in the matrix fraction.
Mitochondrial subfractionation was checked by
immunochemical detection of marker proteins of the
outer membrane (porin), inner membrane (core II
subunit of the cytochrome bc
1
complex), and matrix
(cyclophilin D) (Fig. 3).
Imported CREB promotes expression of
mitochondrial genes
The impact of imported CREB and PKA on the
expression of mitochondrial genes was studied by
testing their effect on the synthesis of [
35
S]methionine-
labelled mitochondrially encoded subunits of oxidative
phosphorylation complexes in rat liver mitochondria.
Addition of cAMP or dibutyryl cAMP (db-cAMP)
A
B
Fig. 2. Inhibition of [
35
S]methionine-labelled CREB mitochondrial
import by proteolytic digestion of mitochondrial outer surface com-
ponents and by an antibody against Tom20. (A) Lane 1: control
import of [
35
S]methionine-labelled CREB. Lane 2: import of the
[
35

S]methonine-labelled CREB followed by trypsin treatment. Lanes
3 and 4: CREB import in mitochondria pretreated for 35 min at 0 °C
with trypsin. Where indicated, mitochondria were also treated with
trypsin after completion of the import incubation. (B) Lanes 1 and
2: as in (A). Lanes 3 and 4: mitochondrial import in the presence of
3 lg of the antibody against Tom70 (Santa Cruz Biotechnology, CA,
USA). Lanes 5 and 6: mitochondrial import in the presence of the
antibody against Tom20 (Santa Cruz Biotechnology). Where indi-
cated, mitochondria were treated with trypsin after completion of
the import incubation. Aliquots of the samples were blotted with
an antibody against the 39 kDa subunit of complex I. See Experi-
mental procedures and [24] for further details.
Fig. 3. Submitochondrial localization of imported [
35
S]methionine-
labelled CREB. Mitochondrial import of [
35
S]methionine-labelled
CREB was followed for 60 min as described in Experimental proce-
dures and in the legend to Fig. 1. After import of [
35
S]methionine-
labelled CREB, followed by trypsin treatment of mitochondria (Mt),
these were spun down and treated to separate mitoplasts (Mp) and
inner membrane (I.M.) and matrix (M.) fractions as described in
Experimental procedures. Aliquots of the samples were analysed by
SDS ⁄ PAGE and autoradiography, or immunoblotted with the anti-
bodies specified. Lanes 1 and 2: mitochondria isolated from the
import mixture, before or after trypsin treatment, respectively. Lane
3: mitoplast fraction. Lane 4: mitoplast fraction subjected to trypsin

treatment. Lane 5: inner membrane fraction. Lane 6: matrix fraction.
For other details, see Experimental procedures.
D. De Rasmo et al. CREB and mitochondrial protein synthesis
FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4327
caused some stimulation of the overall radioactivity
of the gel lane with the [
35
S]methionine-labelled mi-
tochondrially encoded proteins (Fig. 4). In particular
db-cAMP increased the synthesis of ND1 by 80%,
of CoxIII ⁄ ATP6 by 47%, and of ND6 by 30%
(Fig. 4). The addition of the RRL-synthesized CREB
alone resulted in enhancement of the synthesis of
mitochondrial proteins, this effect being strongly
potentiated when CREB was added together with
cAMP, db-cAMP or the catalytic subunit of PKA
(cPKA), respectively, in the incubation mixture
(Fig. 4B, whole gel lane radioactivity of the
[
35
S]methionine-labelled mitochondrial-encoded pro-
teins). In particular, the synthesis of ND1, ND6 and
CoxIII ⁄ ATP6, was enhanced approximately two-fold
by the combination of CREB and cAMP or cPKA
A
B
200
250
125
0

250
125
0
250
125
0
200
400
0
100
0
200
100
0
Fig. 4. Effect of CREB, cAMP and cPKA on mitochondrial protein synthesis. Mitochondrial protein synthesis was performed in a rat liver
mitochondria suspension in the presence of [
35
S]methionine and cycloheximide plus the RRL mixture without the addition of the cDNA
CREB construct (lanes 1–4), or the RRL mixture with the cDNA CREB construct and cold methionine (lanes 5–9). The mitochondrial protein
synthesis control (CTRL) contained: rat liver mitochondria suspension and the RRL mixture without the addition of the cDNA CREB con-
struct. (A) SDS ⁄ PAGE autoradiography of [
35
S]methionine-labelled mitochondrial proteins. Lane 1: control. Lane 2: 50 lM cAMP plus 50 lM
IBMX. Lane 3: 50 lM db-cAMP plus IBMX. Lane 4: cPKA (1 Unit per 10 lg of mitochondrial protein). Lane 5: no addition. Lane 6: cAMP plus
IBMX. Lane 7: db-cAMP plus IBMX. Lane 8: cPKA. Lane 9: CAP (3 mgÆmL
)1
). (B) Histograms showing the mean ADU (as percentage of con-
trol) of the whole gel lane radioactivity of the [
35
S]methionine-labelled mitochondrial proteins and of individual protein spot radioactivity. Mean

values of three separate experiments; **P < 0.01; *P < 0.05. The inset shows autoradiography of [
35
S]methionine-labelled CREB immuno-
precipitated by an antibody against phospho-CREB (Santa Cruz Biotechnology). [
35
S]Methionine-labelled CREB was synthesized in the RRL
translation system in the absence (control) or in the presence of 10 Units of cPKA. Translation products were immunoprecipitated by the
phospho-CREB antibody. For other details, see Experimental procedures.
CREB and mitochondrial protein synthesis D. De Rasmo et al.
4328 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS
(Fig. 4B). Evidence that added cPKA catalysed the
phosphorylation of CREB is provided by a control
experiment showing that the radioactive CREB was
immunoprecipitated by an antibody against phospho-
CREB only when cPKA was added to the mixture
(Fig. 4, inset).
It may be noted that in the experiment presented in
the Fig. 4, not all of the mitochondrially encoded
subunits of the oxidative phosphorylation complexes
exhibited labelling by [
35
S]methionine under the experi-
mental conditions used. The experiment presented in
Fig. 5, in which a higher amount of [
35
S]methionine
was used in the mitochondrial protein synthesis and a
different acrylamide gel concentration was applied for
SDS ⁄ PAGE, shows more subunits of oxidative phos-
phorylation complexes labelled with [

35
S]methionine.
The combined addition of CREB and cAMP or cPKA
resulted also, in this case, in a marked enhancement of
the overall gel lane radioactivity with the [
35
S]methio-
nine-labelled mitochondrially encoded proteins
(Fig. 5B). The autoradiogram presented in Fig. 5A
shows that the synthesis of individual mitochondrially
encoded proteins was generally promoted by the addi-
tion of CREB with cAMP or cPKA. This stimulatory
effect on mitochondrially encoded subunits was com-
pletely abolished by the addition of H89, a specific
inhibitor of PKA.
The addition to the mitochondrial protein synthesis
mixture of the RRL-synthesized NDUFS4 nuclear sub-
unit of complex I, used as a control, had no effect on
the synthesis of mitochondrially encoded subunits of
this and other oxidative phosphorylation complexes
(results not shown). Evidence has been presented else-
where that PKA-mediated phosphorylation of the
NDUFS4 nuclear subunit of complex I [24], as well as
of other nuclear-encoded mitochondrial proteins
[25–27], promotes the import into mitochondria of
these proteins. The presence of cPKA or cAMP in the
import mixture had, however, no effect on the uptake
of CREB by isolated mitochondria (results not
shown).
Discussion

The present results show that in vitro synthesized
CREB is imported into the mitochondrial matrix by a
membrane potential-dependent mechanism. The CREB
imported into mitochondria, and therefore resistant
to digestion by trypsin unless mitochondria were
dissolved by Triton, did not undergo N-terminal pro-
cessing as has also been observed for other nuclear-
encoded mitochondrial matrix-targeted proteins
[26,28]. The inhibition of both mitochondrial surface
binding and import into mitochondria of radioactive
CREB by the antibody against Tom20 shows, how-
ever, that the import of CREB is mediated by the
TOM complex involved in the translocation of pro-
teins into the matrix space [23,29–31]. The antibody
against Tom70, which is an import receptor for inser-
tion of hydrophobic imported proteins into the inner
membrane, did not reduce the import of CREB. The
CREB mitochondrial import could also be assisted by
chaperones, such as the mitochondrial heat shock
protein 70 [19].
A
B
Fig. 5. Effect of H89 on the promotion of mitochondrial protein
synthesis by CREB plus cAMP or cPKA. Mitochondrial protein syn-
thesis was carried out in the presence of the RRL system supple-
mented with cold synthesized CREB, as described in the legend to
Fig. 4. The RRL mixture with cold synthesized CREB was present
in all of the lanes, including the control. The mitochondrial protein
synthesis control (CTRL) contained rat liver mitochondria suspen-
sion and the RRL mixture with the addition of the cDNA CREB con-

struct. (A) SDS ⁄ PAGE autoradiography of mitochondrial proteins
synthesized in the presence of [
35
S]methionine. For the experimen-
tal conditions, see legend to Fig. 4. Where indicated, H89 (100 n
M)
was present during the import incubation. (B) Histograms showing
the mean ADU (as percentage of control) of the whole gel lane
radioactivity of the [
35
S]methionine-labelled mitochondrial proteins.
For other details, see Experimental procedures.
D. De Rasmo et al. CREB and mitochondrial protein synthesis
FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS 4329
Externally synthesized CREB, once imported into
mitochondria, strongly stimulates the synthesis of
subunits of oxidative phosphorylation encoded by
mitochondrial genes; this requires, as in the case of
nuclear CRE-regulated genes, CREB phosphorylation
by PKA [2–4]. The synthesis of all of the mitochondri-
ally encoded subunits of oxidative phosphorylation
enzyme complexes was, in fact, stimulated by the
combined addition of CREB and cAMP or cPKA.
The stimulatory effect was particularly evident
(approximately two-fold enhancement) in the case of
those proteins that were more heavily labelled with
[
35
S]methionine, such as ND1, ND6, and CoxIII ⁄ ATP6
(Figs 4 and 5).

Phosphorylation of CREB, which is synthesized on
cytosolic ribosomes, can take place in vivo in the cytosol
before its import into mitochondria, and ⁄ or after it is
imported into mitochondria. PKA is, in fact, present in
various subcellular regions, including the cytosol and
outer and inner mitochondrial compartments [21,32,33].
Our results show that, under the experimental condi-
tions used, CREB was phosphorylated by the PKA that
was evidently present in the RRL system used for the
in vitro synthesis of CREB and ⁄ or in the mitochondrial
sample, as well as by the added catalytic subunit of
PKA. In this last case, no cAMP was obviously
required. The minor promoting effect on mitochondrial
protein synthesis given by the addition of cAMP or
cPKA (in separate samples) in the absence of added
CREB results from phosphorylation of CREB present
in the RRL system and ⁄ or in the mitochondrial sample
[18–20]. Evidence has been produced showing the exis-
tence of a pool of PKA and PKA-anchoring protein
(AKAP) localized in the inner mitochondrial compart-
ment [21]. Intramitochondrial PKA can be activated by
cAMP generated within mitochondria by a carbon
dioxide ⁄ bicarbonate-regulated soluble adenylyl cyclase
[34,35]. Lee et al. [19] have shown that disruption of
CREB activity, by overexpression of a mito-tagged
negative dominant CREB, decreases the expression of
mitochondrial genes in transfected cells. It has also been
shown that activation of mitochondrial PKA by the
antioxidant deferoxamine results in phosphorylation of
mitochondrial CREB and its binding to the CRE

sequence in the mitochondrial D-loop DNA [20].
In conclusion, the present findings provide unequiv-
ocal evidence that the transcription factor CREB is
imported into the mitochondrial matrix and promotes,
when phosphorylated by PKA, the synthesis of mito-
chondrially encoded subunits of oxidative phosphory-
lation complexes. Our results also lend support, free
from the uncertainties involved in immunochemical
analysis [22], for the presence of CREB in the inner
mitochondrial compartment, where it can also be
phosphorylated by PKA present in the same compart-
ment [21]. This is not a surprise, as CREB, in order to
exert its effect on mitochondrial protein synthesis, has
to move from the cytosol, where it is synthesized,
into mitochondria, where transcription ⁄ translation of
mtDNA-encoded proteins takes place.
Positive modulation by CREB of the expression of
nuclear [36,37] and mitochondrial genes of proteins of
the oxidative phosphorylation system could represent an
important regulatory mechanism for the expression of
this housekeeping cellular function, thus contributing to
the role of CREB in a variety of cellular processes.
Experimental procedures
cDNA construct and in vitro translation
Full-length human CREB cDNA was generated by
RT-PCR, using RNA extracted from primary fibroblasts
from skin biopsy specimens of control subjects. The CREB
cDNA was cloned in the pGEM vector with the T7
promoter. Plasmid construction was confirmed by DNA
sequencing. In vitro transcription ⁄ translation of CREB

cDNA was performed in RRL system (Promega Biotech,
Madison, WI, USA) as reported by De Rasmo et al. [24].
One microgram of CREB construct was added to 50 lLof
Promega standard mixture, containing T7 RNA polymerase
and a standard amino acid mixture with [
35
S]methionine
(20 lCi). Incubation was performed at 30 °C for 90 min.
Rat liver mitochondria
Mitochondria were isolated from rat liver as described in
ref. [38].
Import assay
The assay was performed as in [24]. Sixteen microlitres of the
RRL translation mixture producing [
35
S]methionine-labelled
CREB was added to the import mixture containing
210 mm mannitol, 7 mm Hepes (pH 7.4), 0.35 mm MgCl
2
,
2.5 mgÆmL
)1
BSA, rat liver mitochondria (500 lg of pro-
tein), 3 mm ATP, 3 mm GTP, 15 mm malate and 30 mm
pyruvate in a final volume 200 lL. After incubation at 30 °C
for the times specified in the figures, aliquots of the mixture
were transferred to ice-cooled tubes and supplemented with
protease inhibitors (Sigma, St Louis, MO, USA) (1 lL per
250 lg of mitochondrial protein). Mitochondria were spun
down at 4000 g for 10 min, and supernatant and mitochon-

drial proteins were separated by SDS ⁄ PAGE and transferred
to a nitrocellulose membrane. Radioactive protein bands
were detected by personal fx at phosphorus imager
(Bio-Rad, Milan, Italy) and quantified by versadoc (Bio-
CREB and mitochondrial protein synthesis D. De Rasmo et al.
4330 FEBS Journal 276 (2009) 4325–4333 ª 2009 The Authors Journal compilation ª 2009 FEBS
Rad). The same samples were also immunoblotted with an
antibody against the 39 kDa subunit of complex I of the
respiratory chain (Invitrogen, Paisley, UK).
Mitochondrial protein synthesis
RRL translation medium with unlabelled synthesized
CREB or without CREB was added to the import mixture
containing a standard amino acid mixture with [
35
S]methio-
nine (20 lCi), 210 mm mannitol, 7 mm Hepes (pH 7.4),
0.35 mm MgCl
2
, 2.5 mgÆmL
)1
BSA, rat liver mitochondria
(125 lg of protein), 3 mm ATP, 3 mm GTP, 15 mm malate,
30 mm pyruvate and 50 ngÆmL
)1
cycloheximide in a final
volume of 50 lL. Incubation was performed at 30 °C for
1 h in the presence, where indicated, of cAMP, cPKA,
db-cAMP, isobutylmethylxanthine (IBMX), H89, or
chloramphenicol (CAP). The incubation was prolonged for
10 min after the addition of unlabelled amino acid mixture.

Mitochondria were then spun down at 4000 g for 10 min,
and proteins were separated by SDS ⁄ PAGE and transferred
to a nitrocellulose membrane. Radioactive protein bands
were detected by personal fx at phosphorus imager
(Bio-Rad) and quantified by versadoc (Bio-Rad).
Submitochondrial localization of imported
[
35
S]methionine-labelled CREB
Mitochondrial sublocalization of imported [
35
S]methionine-
labelled CREB was performed essentially as described in
ref. [39]. After mitochondrial import of [
35
S]methionine-
labelled CREB and trypsin treatment, reisolated mitochon-
dria were split in two aliquots and resuspended in 250 mm
sucrose, 1 mm EDTA, and 10 mm Mops ⁄ KOH (pH 7.2),
or in 1 mm EDTA and 10 mm Mops ⁄ KOH (pH 7.2); the
latter medium was used to obtain mitoplasts by mitochon-
drial swelling. After 15 min of incubation on ice, each
sample was split into two, and one of these was again
subjected to trypsin treatment. Mitochondrial and mito-
plasts fractions were spun down at 10 000 g for 10 min.
After mitoplast sonication, the sample was centrifuged at
150 000 g for 15 min. The pellet representing the inner
membrane proteins was resuspended in the SDS ⁄ PAGE
loading buffer; the supernatant, representing the matrix
fraction, was treated with trichloroacetic acid, and the pre-

cipitate was resuspended in the SDS ⁄ PAGE loading buffer.
All samples were analysed by SDS ⁄ PAGE and autoradio-
graphy, or immunoblotted with antibodies against the
porin, cyclophilin D and core II subunit of complex III
(Invitrogen) as specified in the legend to Fig. 3.
Acknowledgements
This work was supported by the National Project on
‘Molecular Mechanisms, Physiology and Pathology of
Membrane Bioenergetics System’, 2005, Ministero
dell’Istruzione, dell’Universita
`
e della Ricerca (MIUR),
Italy, the University of Bari, and Research Foundation
Cassa di Risparmio di Puglia.
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